**Detection of Poxvirus Using Transmission Electron Microscopy Techniques During Outbreak in Bay-Winged Cowbird (***Gnorimopsar Chopi***)**

 M.H.B. Catroxo1, A.M.C.R.P.F. Martins1, S. Petrella2 and L. Milanelo3 *1 Biological Institute of São Paulo, São Paulo,SP, 2 Adolfo Lutz, Institute São Paulo, SP, 3Tietê Ecological Park, São Paulo, SP, Brazil* 

## **1. Introduction**

556 A Bird's-Eye View of Veterinary Medicine

Pfleger KD, Kroeger KM, Eidne KA. Receptors for hypothalamic releasing hormones TRH

Sun Y, Lu X, Gershengorn MC. Thyrotropin-releasing hormone receptors -- similarities and

Takata M, Shimada Y, Ikeda A, Sekikawa K. Molecular cloning of bovine Thyrotropin-

van Wijk HJ, Buschbell H, Dibbits B, Liefers SC, Harlizius B, Heuven HC, et al. Variance

Walling GA, Visscher PM, Andersson L, Rothschild MF, Wang L, Moser G, et al. Combined

Wallis M. Molecular evolution of the thyrotrophin-releasing hormone precursor in vertebrates: insights from comparative genomics. J Neuroendocrinol 2010; 22: 608-19. Wimmers K, Murani E, Ponsuksili S, Yerle M, Schellander K. Detection of quantitative trait loci for carcass traits in the pig by using AFLP. Mamm Genome 2002; 13: 206-10. Yerle M, Pinton P, Robic A, Alfonso A, Palvadeau Y, Delcros C, et al. Construction of a

Porterfield SP, White BA. Endocrine Physiology 3rd. Philadelphia, PA: Mosby Inc., 2007. Sato S, Hasebe H, Sato S, Asahi Y, Hayashi T, Kobayashi E, et al. High-resolution physical

Dev Biol 2004; 15: 269-80.

123-127.

content QTL. Anim Genet 2006; 37: 113-20.

differences. J Mol Endocrinol 2003; 30: 87-97.

Cytogenet Cell Genet 1998; 82: 182-8.

quality on SSC4 and SSC11. J Anim Sci 2007; 85: 22-30.

on porcine growth and fatness. Genetics 2000; 155: 1369-78.

and GnRH: oligomerization and interactions with intracellular proteins. Semin Cell

mapping and construction of a porcine contig spanning the intramuscular fat

Releasing Hormone Receptor gene. Journal of Veterinary Medical Science 1998; 60:

component analysis of quantitative trait loci for pork carcass composition and meat

analyses of data from quantitative trait loci mapping studies. Chromosome 4 effects

whole-genome radiation hybrid panel for high-resolution gene mapping in pigs.

*Gnorimopsar chopi* is a bird with blackish plumage, smooth and silky look, found throughout the Brazilian territory, except in the Amazon State. It is one of the singing birds most wanted by illegal trade and is listed as endargered to extinction by the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA). Avian pox or variola avium is a infectious disease of worldwide distribution, that infect poultry, pet and wild birds of many species which result in economic losses to the poultry industry and commercial aviaries (Trypathy et al., 2000; Weli & Tryland, 2011).

Poxvirus infection has been considered one of important extinction factors for the endangered avian species in Hawaii and in North America (Friend & Franson, 1999; Smits et al., 2005; Kim & Tripathy, 2006).

The etiologic agent is a member of the genus *Avipoxvirus*, *Chordopoxvirinae* subfamily, *Poxviridae* family (Van Riper & Forrester, 2007). The genome is linear, double-stranded DNA molecule rariying in size from 260 to 309 kpb and encodes 260 putative genes (Moss, 2007). Virus particles measure 270 x 450 nm and are composed of an electron dense, centrally located core and two lateral bodies that are visible in ultrathin sections. In negative stained preparations the membrane displays an outer coat composed of a random arranjement of tubules (Carter & Cheville, 1981).

The disease is characterized by cutaneous or wet pox, diphteric or wet pox and septicemic forms. The cutaneous form is the most common in Passeriformes. In this form occurs the development of nodular proliferative skin lesions on the unfeathered parts of the body, legs, feet, face at the base of the beak and eyelids. In the diphtheric form, fibronecrotic lesions occur in the membranes of the upper respiratory tract and esophagus. Anorexia, letargy, ruffled plumage, respiratoty distress, sonnolence, cyanosis and death characterize septicemic poxvirus infections (Ritchie et al., 1994).

Detection of Poxvirus Using Transmission Electron

(Watson, 1958; Reinolds, 1963).

microscope, at 80 kV.

**3. Results** 

**2.4 Immunocytochemistry technique** 

characteristic of cutaneous form (fig. 1, arrow).

Microscopy Techniques During Outbreak in Bay-Winged Cowbird (*Gnorimopsar chopi*) 559

Santander, 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

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

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,

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

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

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,

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

(fig. 3, arrow) measuring 360 x 330 nm.

**3.2 Resin embedding technique** 

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

The curse of the disease is influencied by strain, route of infection, and the species of bird (Ensley et al., 1978).

The incubation period of Avipoxvirus is usually less than a week but may be up to 30 days. The morbidity rate during an outbreak may reach 100% (Ritchie et al., 1994).

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

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

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 techniques.
