Figure 4. Results from necropsy of Tonkean macaque (Macaca tonkeana) from animal sanctuary, Italy, January 2015. A) Hematoxylin and eosin stain of cutaneous lesion. Focal epidermal necrosis, acanthosis ballooning degeneration, and acantholysis of keratinocytes was observed. Staining shows early vesiculation with eosinophilic intracytoplasmic inclusion bodies (arrows) in enlarged degenerated cells. B) Electron micrograph of skin lesion sample showing negatively stained brick-shaped viral particle of ≈160–220 nm, consistent with orthopoxvirus. Scale bar = 100 nm. C) SYBR Green (ThermoFisher Scientific, Waltham, MA, USA) real-time PCR melting curve of all tested samples. The y-axis shows the ratio of the change in fluorescence over the change in temperature. The average melting temperature (80.8°C ± 1°C) was consistent with that for the orthopoxvirus genome.