Lethal Factor Toxemia and Anti-Protective Antigen Antibody Activity in Naturally Acquired Cutaneous Anthrax

Cutaneous anthrax outbreaks occurred in Bangladesh from August to October 2009. As part of the epidemiological response and to confirm anthrax diagnoses, serum samples were collected from suspected case patients with observed cutaneous lesions. Anthrax lethal factor (LF), anti-protective antigen (anti-PA) immunoglobulin G (IgG), and anthrax lethal toxin neutralization activity (TNA) levels were determined in acute and convalescent serum of 26 case patients with suspected cutaneous anthrax from the first and largest of these outbreaks. LF (0.005–1.264 ng/mL) was detected in acute serum from 18 of 26 individuals. Anti-PA IgG and TNA were detected in sera from the same 18 individuals and ranged from 10.0 to 679.5 μg/mL and 27 to 593 units, respectively. Seroconversion to serum anti-PA and TNA was found only in case patients with measurable toxemia. This is the first report of quantitative analysis of serum LF in cutaneous anthrax and the first to associate acute stage toxemia with subsequent antitoxin antibody responses

Debridement increases survival in a mouse model of subcutaneous anthrax.

Anthrax is caused by infection with Bacillus anthracis, a spore-forming gram-positive bacterium. A major virulence factor for B. anthracis is an immunomodulatory tripartite exotoxin that has been reported to alter immune cell chemotaxis and activation. It has been proposed that B. anthracis infections initiate through entry of spores into the regional draining lymph nodes where they germinate, grow, and disseminate systemically via the efferent lymphatics. If this model holds true, it would be predicted that surgical removal of infected tissues, debridement, would have little effect on the systemic dissemination of bacteria. This model was tested through the development of a mouse debridement model. It was found that removal of the site of subcutaneous infection in the ear increased the likelihood of survival and reduced the quantity of spores in the draining cervical lymph nodes (cLN). At the time of debridement 12 hours post-injection measurable levels of exotoxins were present in the ear, cLN, and serum, yet leukocytes within the cLN were activated; countering the concept that exotoxins inhibit the early inflammatory response to promote bacterial growth. We conclude that the initial entry of spores into the draining lymph node of cutaneous infections alone is not sufficient to cause systemic disease and that debridement should be considered as an adjunct to antibiotic therapy

Quantitative Mass Spectrometry for Bacterial Protein Toxins — A Sensitive, Specific, High-Throughput Tool for Detection and Diagnosis

Matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) is a valuable high-throughput tool for peptide analysis. Liquid chromatography electrospray ionization (LC-ESI) tandem-MS provides sensitive and specific quantification of small molecules and peptides. The high analytic power of MS coupled with high-specificity substrates is ideally suited for detection and quantification of bacterial enzymatic activities. As specific examples of the MS applications in disease diagnosis and select agent detection, we describe recent advances in the analyses of two high profile protein toxin groups, the Bacillus anthracis toxins and the Clostridium botulinum neurotoxins. The two binary toxins produced by B. anthracis consist of protective antigen (PA) which combines with lethal factor (LF) and edema factor (EF), forming lethal toxin and edema toxin respectively. LF is a zinc-dependent endoprotease which hydrolyzes specific proteins involved in inflammation and immunity. EF is an adenylyl cyclase which converts ATP to cyclic-AMP. Toxin-specific enzyme activity for a strategically designed substrate, amplifies reaction products which are detected by MALDI-TOF-MS and LC-ESI-MS/MS. Pre-concentration/purification with toxin specific monoclonal antibodies provides additional specificity. These combined technologies have achieved high specificity, ultrasensitive detection and quantification of the anthrax toxins. We also describe potential applications to diseases of high public health impact, including Clostridium difficile glucosylating toxins and the Bordetella pertussis adenylyl cyclase

Debridement significantly increases mouse survival from subcutaneous <i>B. anthracis</i> infection.

<p>(A) Black and white photos of a single A/J mouse infected with 1×10⁵ BIG23 spores in the left ear overlaid with false-color representation of photon emission intensity as indicated by the scale on the right in p/s/cm-2/sr. At 12 hours post injection luminescent infected tissue (left photo- before) was removed (debrided) to eliminate all bacterial luminescence (right photo- after). (B to D) BIG23 spores were injected subcutaneously into the left ear of 4–12 week old A/J mice. Control mice either had their left ear unperturbed (control) or non infected tissue on the same ear was removed (cut control). Mice were then monitored for infection and dissemination using in vivo bioluminescent imaging. Mice were monitored for a total of ten days post-infection (p.i.), until luminescence was detected in a major organ, or mouse appeared moribund (indicating imminent death). (B) Mice were inoculated with 1×10⁵ spores of BIG23. Luminescent tissue was debrided at 12 hours p.i. Survival data from a total of seven mice from three independent experiments were analyzed with the log rank test to determine significant differences in survival between debrided and control mice (*, p = 0.0108), and debrided and cut control mice (**, p = 0.0021). There was no statistical difference between control and cut control mice. (C) Mice were inoculated with 1×10⁶ spores of BIG23. Luminescent tissue was debrided at 12 hours p.i. Survival data was analyzed with the log rank test on a total of ten debrided mice and seven mice each for the control groups from three independent experiments to determine significant differences in survival between debrided and control mice (*, p = 0.0109) and debrided and cut control mice (**, p = 0.0026). There was no statistical difference between control and cut control mice. (D) Mice were infected with BIG23 as described above, but debridement of luminescent tissue was performed at 24, 48, or 78 hour p.i. Survival data was analyzed with the log rank test on a total of seven mice from each experimental group, with the exception of the control mice that totaled six, from three independent experiments to determine significant differences from control mice. The survival of mice debrided at 24 hours (*, p = 0.0110) and 48 hours (*, p = 0.0477) p.i. was significantly increased. The survival of mice debrided at 72 hours p.i. was not significantly different than control mice (p = 0.1734).</p

Presence of lethal factor in cervical draining lymph node does not alter cellular activation.

<p>Mice were infected with 1×10⁶ Sterne strain or TKO spores subcutaneously in the left ear. At 24 hours cLN were taken from mice including: Sterne infected mice (solid line non-shaded) along with control non-infected contralateral lymph nodes from Sterne infected mice (solid line shaded), and TKO infected (dashed line non-shaded) and control non-infected contralateral lymph nodes from TKO infected mice (dashed line shaded). cLN were homogenized and cells were stained for flow cytometry. Flow plots were gated for single event live CD45 positive cells. (A) Left- a representative histogram comparing expression of CD69 in draining cLN. Right- The mean fluorescence intensity of lymph nodes draining either Sterne (*, p = 0.0464) or TKO (*, p = 0.0256) infections from seven mice was significantly higher than contralateral control lymph nodes as determined using Student's T test. (B) Left- a representative histogram comparing expression of CD86 in draining cLN. Right- The mean fluorescence intensity of lymph nodes draining either Sterne (*, p = 0.0119) or TKO (**, p = 0.0063) infections from seven mice was significantly higher than contralateral control lymph nodes as determined using Student's T test. (C) A representative comparison of CD80 expression in draining cLN. The mean fluorescence intensity of lymph nodes draining either Sterne or TKO infections from seven mice was not significantly higher than lymph nodes that were not.</p

Anthrax-like cutaneous lesion on left check of 70-year-old male Florida resident.

<p>Anthrax-like cutaneous lesion on left check of 70-year-old male Florida resident.</p

Results of serological testing of specimens from 2013 Florida patient with anthrax-like cutaneous lesion.

<p>Results of serological testing of specimens from 2013 Florida patient with anthrax-like cutaneous lesion.</p

<i>B</i>. <i>cereus</i> BcFL2013 capsule. India ink stain of <i>B</i>. <i>cereus</i> BcFL2013 after overnight growth in defibrinated horse blood.

<p><i>B</i>. <i>cereus</i> BcFL2013 capsule. India ink stain of <i>B</i>. <i>cereus</i> BcFL2013 after overnight growth in defibrinated horse blood.</p