**3. Results**

558 A Bird's-Eye View of Veterinary Medicine

The virus may be mechanically transmited by insect vectors such as mosquitoes, mites or ticks, by direct contact with another infected bird, by contact with contamined food, water,

The curse of the disease is influencied by strain, route of infection, and the species of bird

The incubation period of Avipoxvirus is usually less than a week but may be up to 30 days.

Transmission electron microscopy is a classic tool for the diagnosis of poxviruses, where the viral particles with characteristic morphology are present in large numbers in swabs, biopsies or dry crusts (OIE, 2008). This method has typically been used by national reference

The aim of this study was to detect the presence of avianpoxvírus particles in samples of skin lesions of *Gnorimopsar chopi* using negative staining (rapid preparation) immunocytochemistry (immunolabeling with colloidal gold particles) and resin embedding

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

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,

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 (González-

The morbidity rate during an outbreak may reach 100% (Ritchie et al., 1994).

or research laboratories to identify avianpoxvirus (Weli et al., 2004).

Non-specific stress factors are associated with viral reactivation (Ritchie et al., 1994).

semen or surfaces (Metz et al., 1985; Ritchie & Carter, 1995).

(Ensley et al., 1978).

techniques.

**2. Material and methods** 

**2.1 Description of the outbreak** 

immunocytochemistry techniques.

**2.3 Resin embedding technique** 

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

pH 5.0 (Brenner & Horne, 1959; Hayat & Miller, 1990; Madeley, 1997).

Among the 45 analyzed animals, all of them (100%) presented in feet, and occasionally in beaks and wings, small yellowish-brown proliferative nodules or scabs of different sizes, characteristic of cutaneous form (fig. 1, arrow).

Fig. 1. Nodular and crusted lesions on the feet of *Gnorimopsar chopi* (arrow).

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

On the transmission electron microscopy by the negative staining technique, two types of poxvirus particles were visualized in all the analyzed samples. the M form, with regular spaced thread-like ridges comprising the exposed surface (fig. 2, big arrow), measuring 280 x 230 nm and the C form or stain-penetrated particle showing the dumbbell-shaped core (fig. 3, arrow) measuring 360 x 330 nm.

#### **3.2 Resin embedding technique**

Using the resin embedding technique (positive staining) were visualized in the ultrathin sections, three types of intracytoplasmic inclusion bodies. The type A or Bollinger body (fig. 4), outlined by membrane, containing in its interior a great number of mature particles (fig. 4,

Detection of Poxvirus Using Transmission Electron

Microscopy Techniques During Outbreak in Bay-Winged Cowbird (*Gnorimopsar chopi*) 561

Fig. 4. Ultrathin sections of the scabs fragments showing type A or Bollinger

intracytoplasmic inclusion bodies, containing mature particles (arrow). Bar: 1040 nm.

Fig. 5. Ultrathin sections of the skin lesions fragments, showing mature particles with inner dumbell shaped core (big arrow), lateral bodies (minor arrow) and external envelope (blue

arrow). Bar: 280 nm.

Fig. 2. Negatively stained avian poxvirus particles showing the M form, with regular spaced thread-like ridges comprising the exposed surface (arrow). Bar: 180 nm.

Fig. 3. Negatively stained avian poxvirus particles showing the C form or stain-penetred particle reveling the dumbell-shaped core (arrow). Bar: 200 nm.

arrow), measuring 200 x 300 nm, revealing the inner dumbbell-shaped core (fig. 5, big arrow), two lateral bodies (fig. 5 minor arrow) and an external envelope (fig. 5, blue arrow). In the type B electron dense inclusions bodies, viral particles budding of dense amorphous material were observed (figs. 6 and 7, arrow). Fibrillar inclusions constituted by groups of fibrils (fig. 8, arrow) were disposed in groups varying from 2 up to 7 and sometimes showed cross striations.

Fig. 2. Negatively stained avian poxvirus particles showing the M form, with regular spaced thread-like ridges comprising the exposed surface (arrow). Bar: 180 nm.

Fig. 3. Negatively stained avian poxvirus particles showing the C form or stain-penetred

arrow), measuring 200 x 300 nm, revealing the inner dumbbell-shaped core (fig. 5, big arrow), two lateral bodies (fig. 5 minor arrow) and an external envelope (fig. 5, blue arrow). In the type B electron dense inclusions bodies, viral particles budding of dense amorphous material were observed (figs. 6 and 7, arrow). Fibrillar inclusions constituted by groups of fibrils (fig. 8, arrow) were disposed in groups varying from 2 up to 7 and sometimes showed

particle reveling the dumbell-shaped core (arrow). Bar: 200 nm.

cross striations.

Fig. 4. Ultrathin sections of the scabs fragments showing type A or Bollinger intracytoplasmic inclusion bodies, containing mature particles (arrow). Bar: 1040 nm.

Fig. 5. Ultrathin sections of the skin lesions fragments, showing mature particles with inner dumbell shaped core (big arrow), lateral bodies (minor arrow) and external envelope (blue arrow). Bar: 280 nm.

Detection of Poxvirus Using Transmission Electron

groups of fibrils or lamelae (arrow). Bar: 320 nm.

1980; Van Riper et al., 2002; Catroxo et al., 2009).

130 nm.

Microscopy Techniques During Outbreak in Bay-Winged Cowbird (*Gnorimopsar chopi*) 563

Fig. 8. Ultrathin sections of the skin fragments revealing fibrillar inclusions constituted by

Fig. 9. In the immunocitoquemistry technique, the antigen-antibody interaction was strongly enhanced by the dense colloidal gold particles (arrow) over the avipoxvirus particles. Bar:

Lesions similar have been described in other birds (Goodpasture & Anderson, 1961; Trapp,

Lesions can interfere wich perching when occur on the feet or legs and frequently lead to

Clinical signs of lack appetite, weight lost, difficulties of locomotion, diarrhea, dehidratation and death were observed in all birds of this study. This has been documented in similar occurrence of avianpox in other avian species (Mc Donald et al., 1981; Docherty et al., 1991;

Ostrowski et al.., 1995; Gerlach et al., 1998; Kreuder et al., 1999; Catroxo et al., 2009).

development of secondary infections (Van Riper III & Forrester, 2007).

Fig. 6. Ultrathin sections of the scabs fragments reveling type B electron dense inclusions bodies with viral particles budding of dense amorphous material (arrow). Fig. 6 - Bar: 600 nm.

Fig. 7. Bar: 430 nm.

#### **3.3 Immunocytochemistry technique**

In the immunocytochemistry technique, the antigen-antibody interaction was strongly enhanced by the dense gold particles over the viruses (Fig. 9, arrow).
