**2.1 Description of the outbreak**

In the period of the 2005 to 2007, during illegal commercialization of Brazilian birds, 60 baywinged cowbird (*Gnorimopsar chopi*) were apprehended and being forwarded to the CRAS (Wild Animals Recovery Center, Ecological Park of Tietê). After one or two months, 45 birds presented cutaneous lesions in the feet. Some of them also had lesions on the beak and wings. The animals also been presented clinical signs of anorexia, emaciation, locomotion difficulties, diarrhea, dehydration and death. The birds were sent to the Electron Microscopy Laboratory of the Biology Institute of São Paulo to investigate viral agents. Scabs and fragments of skin lesions collected of these birds were processed for transmission electron microscopy utilizing negative staining (rapid preparation), resin embedding and immunocytochemistry techniques.

#### **2.2 Negative staining technique (rapid preparation)**

In the negative staining the scabs and fragments of skin lesions were suspended in phosphate buffer 0.1 M, pH 7.0. Drops of the obtained suspension were placed in contact with metallic copper grids with carbon stabilized supporting film of 0.5% collodium in amyl acetate. Next, the grids were drained with filter paper and negatively stained at 2% ammonium molybdate, pH 5.0 (Brenner & Horne, 1959; Hayat & Miller, 1990; Madeley, 1997).

#### **2.3 Resin embedding technique**

Thin slices of scabs and fragments of skin lesions were fixed in 2.5% glutaraldehyde in 0.1M, pH7.0 phosphate buffer and pos-fixed in 1% osmium tetroxide in the same buffer. After dehydration in cetonic series, the fragments were embedded in Spurr resin (GonzálezSantander, 1969; Luft, 1961). Ultrathin sections were cut on the LKB ultratome and mounted on copper grids. The sections were stained by combination of uranyl acetate-lead citrate (Watson, 1958; Reinolds, 1963).

## **2.4 Immunocytochemistry technique**

At the immunolabeling technique with colloidal gold particles for negative staining, the copper grids were placed in contact with viral suspension and, after removing excess with filter paper, the same were put on specific primary antibody drops. After successive washings in PBS drops, the grids were incubated in protein A drops in association with 10 nm gold particles (secondary antibody). Grids were then contrasted at 2% ammonium molybdate, pH 5.0 (Knutton, 1995). Observations were made in a Philips EM 208 electron microscope, at 80 kV.
