Primary Involvement of Pharynx and Peyer's Patch in Inhalational and Intestinal Anthrax

Abstract

Bacillus anthracis causes three forms of anthrax: inhalational, gastrointestinal, and cutaneous. Anthrax is characterized by both toxemia, which is caused by secretion of immunomodulating toxins (lethal toxin and edema toxin), and septicemia, which is associated with bacterial encapsulation. Here we report that, contrary to the current view of B. anthracis pathogenesis, B. anthracis spores germinate and establish infections at the initial site of inoculation in both inhalational and cutaneous infections without needing to be transported to draining lymph nodes, and that inhaled spores establish initial infection in nasal-associated lymphoid tissues. Furthermore, we found that Peyer's patches in the mouse intestine are the primary site of bacterial growth after intragastric inoculation, thus establishing an animal model of gastrointestinal anthrax. All routes of infection progressed to the draining lymph nodes, spleen, lungs, and ultimately the blood. These discoveries were made possible through the development of a novel dynamic mouse model of B. anthracis infection using bioluminescent non-toxinogenic capsulated bacteria that can be visualized within the mouse in real-time, and demonstrate the value of in vivo imaging in the analysis of B. anthracis infection. Our data imply that previously unrecognized portals of bacterial entry demand more intensive investigation, and will significantly transform the current perception of inhalational, gastrointestinal, and cutaneous B. anthracis pathogenesis.

Noninvasive Imaging Technologies Reveal Edema Toxin as a Key Virulence Factor in Anthrax

“…Production of LT by the 9602C-lux(LFϩPAϩ) strain led to a progression of infection similar to that previously reported for the nontoxinogenic encapsulated 9602P-lux(ToxϪ) 8 ( Figure 2, A and E). Bioluminescence was observed in the ear within 21 hours after inoculation ( Figure 2A); dissemination to the draining cervical lymph node occurred within 26 hours in 89% of the mice and systemic dissemination to the spleen and lungs within 48 Ϯ 4 hours (mean Ϯ SEM, n ϭ 6; range, 45 to 68 hours).…”

“…24,25 Luminescent B. anthracis strains used in this study were constructed by insertion of the luxABCDE operon of Photorhabdus luminescens as described previously. 6,8 Expression of the lux operon did not affect the virulence of the strains, as evidenced by equivalent mean time to death compared with the parental strains (data not shown).…”

“…Mice were challenged with each luciferase-expressing bacterial strain either into the ear pinna (3.40 Ϯ 0.29 log 10 CFU, mean Ϯ SD, in 10 L) or by intranasal route (6.52 Ϯ 0.21 log 10 CFU, mean Ϯ SD, in 20 L) as described previously 7,8 and were analyzed at least twice a day. The challenge inocula were lethal, and all mice displayed bioluminescence on infection.…”

“…Encapsulated bacilli were detected by immunohistochemistry (IHC) using a rabbit polyclonal antiserum specific for capsule polyglutamate as described previously. 8 For detection of apoptosis processes, an in situ cell death detection kit (Roche, Mannheim, Germany) was used, based on labeling of DNA strand breaks (TUNEL technology: terminal deoxynucleotidyl transferase dUTP nick end-labeling), according to the manufacturer's instructions.…”

“…Nevertheless, bacteria could be detected in the cervical lymph node (2.29 Ϯ 0.3 log 10 CFU per lymph node , n ϭ 3) when the spleen displayed bioluminescence; this shows that the bacteria transited through the lymph node, although the bacterial load remained below the detection threshold of the bioluminescence signal. 8 This suggests that ET did not allow extensive local multiplication in the draining lymph node.…”