Debate about the appropriate application of debridement to soft tissue infections by B. anthracis
has been hampered by lack of experimental evidence to indicate or contraindicate its use. We thus sought to develop a mouse model for debridement. To achieve this goal spores from a B. anthracis
Sterne strain that produces light during vegetative growth within a host (BIG23) were injected subcutaneously in the ear of C5-deficient A/J mice. Infection of A/J mice with non-capsulated toxigenic B. anthracis
Sterne strain is a widely used animal model for anthrax that recapitulates many characteristics of infections caused by encapsulated toxigenic B. anthracis
strains such as the Ames strain 
, yet poses less risk to researchers, and thus makes the model described here broadly accessible to researchers without BSL3 facilities. The ear was chosen as the anatomic site of infection for our studies because the ear architecture allows efficient debridement, its translucence permits greater sensitivity when detecting bacterial growth by light emission, and has been frequently used for modeling infectious diseases 
. Bioluminescent B. anthracis
strains were applied in order to qualitatively identify the initial site of infection, determine whether all infected tissues were removed during the debridement procedure, and allow the assessment of the dissemination of bacteria over time. Initial sites of infection were identified by bacterial luminescence at 12, 24, 48, and 72 hours and debrided by notching the ear to efficiently remove the initial site of infection (); similar to general mouse husbandry ear tagging procedures. Control infected mice had a section of ear removed that did not contain the infection, or as a second control, no portion of the ear was removed. Debridement at 12 hours significantly protected mice from subcutaneous infection with two different doses of spores (). All mice that were inoculated with 1×105
spores and debrided survived, whereas 67% of the control groups succumbed to systemic infections (). Similarly, in mice inoculated with 1×106
spores (approximately 100 LD50
s), 50% of the debrided mice survived, while all mice from control groups succumbed to systemic infections (). Debridement of the ear significantly decreased mortality when performed at 12, 24, and 48 hours post-infection, but lost significance after 72 hours post-infection (). We conclude that debridement significantly protected mice from subcutaneous infection if performed within 48 hours of subcutaneous inoculation.
Debridement significantly increases mouse survival from subcutaneous B. anthracis infection.
There are multiple mechanisms by which debridement could promote survival, one of which is the prevention of bacterial dissemination from the site of inoculation to the draining lymph node. We thus questioned the timing of entry of B. anthracis
into the draining lymph node post-inoculation and if bacteria had indeed entered the draining lymph nodes during the interval between inoculation and debridement as is predicted by the Trojan horse model of infection. Accordingly, at 1, 12 and 24 hours post-infection the cervical draining lymph nodes were removed and bacterial colony-forming units (CFU) were measured for both the total number of bacteria (spores+vegetative cells) by plating without heat treatment or for spores-only by heating the samples to 65°C for 20 minutes to kill all germinating spores and vegetative bacteria, and then plating for CFU 
. Independent of the initial dose, approximately 3–4% of injected spores arrived in the draining lymph nodes as early as one hour after injection (). The number of spores in the lymph node remained constant, and no outgrowth occurred as late as 24 hours after infection (). To test the effect of debridement on subsequent bacterial loads, lymph nodes were removed at 24 and 72 hours from mice that underwent debridement at 12 hours or non-debrided control mice. At 72 hours post-injection the CFU had dropped significantly by over a log and as at earlier time points, all CFU were comprised of spores (). With debridement there were significantly fewer spores in the cLN at 24 hours post-infection (p
0.0144), and insignificantly fewer spores at 72 hours post-infection (p
0.0066) (). These findings were unaffected by the dose of spores administered, however the magnitude of measured CFU was proportionally lower with the lower inoculum dose (). At no time were CFU isolated from the contralateral lymph nodes draining the uninoculated right ear (data not shown). Similar cLN CFU were observed in infections using spores from a B. anthracis
strain that had PA, LF, and EF production eliminated (TKO) (), suggesting that the above findings are spore mediated and not LT or ET dependent. In total these data suggest that at the time of debridement at 12 hours post-inoculation spores can be found in the draining lymph nodes and thus these spores are typically not sufficient to cause systemic infections since debridement effectively increased the likelihood of mouse survival.
B. anthracis spores are found in the draining cervical lymph node early in infection.
The clinical difficulty of treating anthrax, even after the bacteria have been effectively controlled with antibiotics, has been attributed to residual exotoxin activity within the host. Likewise, B. anthracis
exotoxins have been reported to modulate chemotaxis and activation of host innate immune cells 
. We thus questioned whether exotoxins were present at the time of debridement, since they could be locally influencing the chemotaxis of potential Trojan horse cells, altering the activation state of innate immune defenses, or contributing to overall mortality even after the bacteria were surgically removed. At 12 hours, the earliest time of debridement, 458±216 pg LF was isolated from the ear, 28±10 pg in the draining lymph node, and 476±82 pg/ml in the serum of mice that received 1×106
BIG23 spores subcutaneously (). When TKO spores were co-inoculated with purified LT (100 ng LF+650 ng PA), the LF distributed into the ear, lymph nodes, and serum similarly to the LF actively produced by the exotoxin-producing BIG23; with relatively equal proportions of the total LF distributed to the ear and serum and approximately one-twentieth of the quantity found in the cLN as in the ear and serum (). These LF concentrations were assessed using a highly sensitive mass spectrometry-based assay for LF enzymatic activity that was originally designed to assess LF concentrations in serum 
, but was adapted here to study solid tissues. To date this is the most sensitive assay for detecting any of the components of the B. anthracis
exotoxins and may reflect total exotoxin production since the individual exotoxin components are coordinately regulated 
. Notably, the majority of debrided mice did not succumb to infection, even with measurable circulating exotoxins, implying that these concentrations of LF alone are not sufficient to mediate death. Likewise when TKO spores were used to initiate infection, a similar number of spores were found in the cLN as wild-type Sterne strain bacteria during the same time period (). This suggests that spore entry into the cLN is not affected by LT or ET, counter to what may have been predicted based on published cell culture analyses that demonstrated exotoxin-based alterations in phagocyte activation and chemotaxis towards lymph nodes 
Lethal Factor is measurable in the ear, draining cervical lymph nodes and serum at the time of debridement 12 hours after spore inoculation.
Since exotoxins were present at the earliest time of debridement, we next asked whether they had any measurable effect on innate immune cell activation within the draining lymph nodes. Accordingly, spores from B. anthracis
Sterne or the TKO mutant were injected subcutaneously into the left ear of mice at a concentration of 1×106
spores and at 24 hours cLN were removed, homogenized, and stained for flow cytometric analysis. Expression of the activation markers CD69, CD80 and CD86 were chosen as measures of activation since previous publications have demonstrated that these cell markers are down regulated by B. anthracis
. Specifically, CD80 and CD86 are down regulated in dendritic cells 
, and CD69 is down regulated in response to LT in T cells at 24 hours 
. Flow cytometry revealed that expression of CD69 and CD86, but not CD80, were significantly up-regulated in mice that had been infected with Sterne or TKO spores as compared to internal control lymph nodes (). The degree of up-regulation was the same regardless of the ability of bacteria to produce exotoxin. cLN analyzed at 12 hours post-infection demonstrated no increase in CD69, CD80, or CD86 under any conditions (data not shown). These findings suggest that exotoxins are not present in the lymph node in sufficient quantities to alter cellular response to inflammatory stimuli in the first 24 hours of B. anthracis
Presence of lethal factor in cervical draining lymph node does not alter cellular activation.