Table 1.
Ziehl-Neelsen staining, culture and PCR results for 5 aquatic specimens collected in Buruli ulcer endemic regions of Benin and Togo
Table 2.
Results of inoculation of mouse footpads with BACTEC cultures of 2 environmental specimens
Table 3.
Phenotypic characteristics of isolate 00-1441 and M. ulcerans geographic subgroups
Figure 1.
Mass spectroscopic analysis of ASL of M. ulcerans 00-1441 showing the hydrolysis product of mycolactone A/B at m/z 447.3 (*).
The intact mycolactone can be identified at lower intensity at m/z 765.9 (**).
Figure 2.
Mass spectroscopic analysis of the ASL of M. ulcerans 00-1441 showing the mycolactone A/B sodium adduct at m/z 765.6.
(In this case an ion trap was set to select for positive ions in the range m/z 755–775.)
Figure 3.
MS/MS fragmentation pattern of the mycolactone A/B ion at m/z765.6.
The two main characteristic fragments are those of the macrolide ring m/z = 429.4 (*) and the side chain at m/z = 359.3 (**).
Figure 4.
Cytotoxic activity of M. ulcerans 00-1441 to BMDM.
BMDM were infected with M. ulcerans 00-1441 at an MOI of 1∶1. Macrophages were photographed by phase-contrast microscopy at 4 h (A), 4 days (B), and 6 days (C) postinfection. Macrophage cell rounding, shrinkage and detachment are present at days 4 and 6 post-infection.
Figure 5.
Footpad swelling and bacterial proliferation during infection with isolate 00-1441.
BALB/c mice were infected subcutaneously in the footpad with 5.2 log10 AFB of M. ulcerans 00-1441. The degree of pathologic changes was assessed by measurement of footpad swelling (▪) (n = 8). The proliferation of bacilli in the footpad was determined by counting AFB (open bars) of footpad homogenates (n = 5). Significant differences were performed using Student's t test (**, p≤0.01).
Figure 6.
Histologic sections of mouse footpads infected with M. ulcerans 00-1441.
BALB/c (A–C) and NMRI (D) mice were infected subcutaneously with 5.4 log10 M. ulcerans 00-1441. At different time points the footpads were harvested and processed for histologic analysis. Dermal edema could be found by the second week of infection (A), along with a necrotic center surrounded by both chronic and acute inflammatory infiltrates (B) (hematoxylin-eosin stained sections). After 4 weeks of infection, bacilli were observed co-localized with cells (C) and in large clumps in the necrotic center of the lesion (D) (Ziehl-Neelsen stained sections).
Figure 7.
Ziehl-Neelsen staining of bone tissue of mice infected with M. ulcerans 00-1441 (A, B and inset of D, arrows).
NMRI mice were infected subcutaneously in the footpad with 5.4 log10 M. ulcerans 00-1441. At 6 weeks post-infection, bacilli were found both in the bone tissue and in the bone marrow (A, B and inset in D, arrows). Hemotoxylin-eosin staining of C and D reveals extensive destruction of bone. Outlines of dead cortical bone spicules are seen in C and D (arrowheads). Marrow is necrotic and replaced by chronic inflammation.
Figure 8.
An example of Afrotropical Gerridae: Limnogonus hypoleucus (Gerstaecker).
Photo: Jérome Constant, Department of Entomology, Royal Belgian Institute of Natural Sciences, Brussels, Belgium.
Figure 9.
Main steps followed to cultivate M. ulcerans in pure culture from an aquatic insect (Gerris sp.).