Figure 1.
Macroscopic appearance of pig skin infected with M. ulcerans.
Development of representative lesions 2.5 weeks or 6.5 weeks after subcutaneous infection with 2×107 (B1, D1 and E1) or 2×106 (C1) CFU is depicted and compared to a site left uninfected (A1). Excised tissue specimens were fixed and vertically cut in half to visualize macroscopically visible alteration in tissue structure (A2–E2). For high inoculation doses (≥2×106 CFU) the formation of nodules (B1) with necrotic centres was observed already 2.5 weeks after infection (B2). These yellow centres indicative for coagulative necrosis were surrounded by a reddish ring (B2). At 6.5 weeks after infection, these nodules had progressed into a small ulcer or a plaque (D1, E1) associated with marked macroscopically visible alterations in tissue structure (D2, E2). At sites injected with 2×106 CFU nodules with greyish discoloration of the dermis had developed 6.5 weeks after injection of M. ulcerans (C1, C2).
Figure 2.
Microscopic appearance of pig skin 2.5 weeks after experimental infection.
Histologic sections stained with HE. Infiltrating cells were found in the fat layer between dermis and muscle tissue at sites infected with ≥2×104 CFU (A1, B1, C1 and D1). While the two highest inoculation doses led to the development of lesions with a necrotic core surrounded by strong infiltration (A2, B2), infiltration but no necrotic core was observed when doses of 2×104 and 2×103 CFU were used (C2, D2 insert). Fat cell ghosts were found at sites infected with the three highest inoculation doses (A2, B2 and C2). x: necrosis, y: infiltration, z: fat cell ghosts.
Figure 3.
Microscopic appearance of pig skin 6.5 weeks after experimental infection.
Histologic sections stained with Haematoxylin/Eosin (HE) (A1, A2, B1, C1, D1 and D2) or Ziehl-Neelsen/Methylene blue (ZN) (B2, C2). At sites injected with 2×107 CFU nodules had either developed into a small ulcer with destroyed epidermis (z), strong infiltration and indications for the development of undermined edges (A1, A2, dotted line) or into a plaque with a necrotic core surrounded by infiltrating cells (B1). x: intact epidermis, y: epidermal hyperplasia, z: destroyed/missing epidermis. The site infected with 2×106 CFU showed a similar architecture as the plaque but flatter, less organized and with a smaller overall circumference (C1). Both lesions comprised AFB in their necrotic cores, either in big clumps (B2) or in smaller numbers and smaller aggregations (C2). No signs of infection, inflammation and pathology were observed at sites inoculated with 2×105 CFU (D1, D2) or less (not shown).
Figure 4.
Containment of large amounts of AFB in the necrotic core and development of satellite microcolonies.
Histologic sections stained with ZN. A plaque (A) and a small ulcer (B) are shown that developed 6.5 weeks after infection with 2×107 CFU. The ulcerated lesion was strongly infiltrated at the site of ulceration, where no AFB were found (Region 1, B1, B2). Lateral and between dermis and muscle tissue infiltrating cells enclosed small necrotic areas (Region 2, B3), where AFB were found as satellite microcolonies (B4, B5). The plaque consisted of distinct layers of infiltrating cells encasing a necrotic core containing large clumps of bacteria (Ring 1, A1, A2). A second and third ring with decreasing bacterial load and integrity and increasing integrity of infiltrating cells were layered around this core (Ring 2, A3, A4 and Ring 3, A5, A6). A belt of intact cells was surrounding these three inner layers. It did not contain any AFB (Ring 4, A7) except for a microcolony peripheral to the main bacterial burden (Ring 5, A8).
Figure 5.
Histophathological hallmarks of Buruli ulcer in experimentally infected pig skin.
Histologic sections stained with Haematoxylin/Eosin (HE) (A1, A2, A5, A6, A7, A8, A9, B1, B2 and B3) or Ziehl-Neelsen/Methylene blue (ZN) (A3, A4). A: All typical histopathological features of BU in humans were found in infected pig skin. A1: necrosis, A2: fat cell ghosts, A3 and A4: extracellular clusters of AFB, A5: healthy epidermis, A6: moderate epidermal hyperplasia, A7: strong epidermal hyperplasia, A8: granuloma formation, A9: giant cells. B: Histopathological changes induced by mycolactone injection. B1: necrosis, B2: fat cell ghosts, B3: giant cells.