Avipoxvirus outbreak in cutaneous lesions of the red-cowled cardinal (Paroaria dominicana) and red-crested cardinal (Paroaria coronata): detection by histopathology and transmission electron microscopy

Authors: Nataly Alves Melo; Ana M Cristina R P F Martins; Mathias Fritzen Pedroso; Marcia Helena Braga Catroxo
Avipoxvirus outbreak in cutaneous lesions of the red-cowled cardinal (Paroaria dominicana) and red-crested cardinal (Paroaria coronata): detection by histopathology and transmission electron microscopy
DOI
10.25125/ijoear-oct-2026-13
DIN
IJOEAR-OCT-2026-13
Abstract

The red-cowled cardinal (Paroaria dominicana) and red-crested cardinal (Paroaria coronata) are birds of the family Emberizidae naturally found in northeastern and southern Brazil, respectively. These species are targeted by illegal trade because of their colorful plumage and are considered potential viral reservoirs owing to their migratory characteristics. Avian pox is a highly infectious disease caused by poxviruses of the family Poxviridae and genus Avipoxvirus that affects domestic and wild birds. Between January 2006 and September 2007, 72 birds, including 14 red-crested cardinals and 58 red-cowled cardinals, were referred after sudden death by CRAS-PET to the Electron Microscopy Laboratory of the Biological Institute of São Paulo for investigation of viral agents. At necropsy, small cutaneous nodular lesions were observed on the feet of one red-crested cardinal and ten red-cowled cardinals. Some birds had lesions characteristic of avian pox on the feet and/or beaks, consisting of whitish or yellowish nodules and desquamating crusts. Lesion fragments were processed for histopathology and transmission electron microscopy. Light microscopy revealed hyperkeratosis, epithelial hyperplasia, hydropic degeneration, and eosinophilic inclusions corresponding to Bollinger bodies in skin and/or mucosal cells. Negative staining identified numerous typical poxvirus particles in ten crust-suspension samples from red-cowled cardinals and one from a red-crested cardinal. Some particles were enveloped and brick-shaped, with irregularly arranged tubules on the outer membrane, and measured 280 × 260 nm. Ultrathin sections revealed type A intracytoplasmic inclusion bodies, or Bollinger bodies, containing mature or complete oval viral particles measuring 330 × 260 nm, with a biconcave nucleus or dumbbell-shaped internal core. Type B inclusion bodies, immature particles budding from dense amorphous filamentous material, developing particles with spikes on their outer surface, fibrillar inclusions, and deformed nuclei with densely condensed chromatin were also identified. Immunocytochemistry with colloidal gold labeled viral particles and antigenic sites in intracytoplasmic vacuoles and cellular debris. This is the first record of avian pox in the red-crested cardinal (Paroaria coronata) and the second in the red-cowled cardinal (Paroaria dominicana).

Keywords
Paroaria coronata; Paroaria dominicana; poxvirus; transmission electron microscopy.
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Introduction

The red-cowled cardinal (Paroaria dominicana) (LINNAEUS, 1758) and the red-crested cardinal (Paroaria coronata) (MULLER, 1776) belong to the order Passeriformes and genus Paroaria. The red-cowled cardinal, also known as cabeça-vermelha or cardeal-do-nordeste, is characteristic of the Caatinga and occurs mainly in northeastern Brazil, although it is also found in southern Bahia and Minas Gerais. The red-crested cardinal occurs in Brazil, Uruguay, Paraguay, Bolivia, and Argentina (SICK, 2001).

Both species are frequently affected by the illegal wildlife trade. The red-cowled cardinal has been identified as one of the most commonly traded species in open-air markets in the Recife Metropolitan Region and as the bird most frequently seized in the Campina Grande region, Paraíba (MENEZES et al., 2003; PEREIRA & BRITO, 2005). Of 1,808 birds seized by the São Paulo Department of Parks and Green Areas, 148 were red-cowled cardinals (P. dominicana) (CAVALHEIRO et al., 2003). Red-crested and red-cowled cardinals are also among the species most commonly seized in the South and Central-West regions, while the red-cowled cardinal is among those most commonly seized in southeastern and northeastern Brazil (IBAMA, 2007).

Wild birds are considered potential viral reservoirs because their migratory behavior allows long-distance movements and dissemination of viruses of epidemiological importance. In addition, stressors such as capture, transport, confinement, and dietary or climatic changes may trigger viral processes, leading to sudden outbreaks and varied clinical manifestations (CUBAS & GODOY, 2004).

Diseases affecting avian skin have gained increasing importance because they can cause reduced egg production and growth, blindness, and, in some cases, death. Although natural avian poxvirus infection is better known in domestic poultry, it has also been reported in free-living birds (FENNER et al., 1992).

Avian pox is caused by viruses of the family Poxviridae and genus Avipoxvirus. Poxviruses have double-stranded DNA genomes 130–375 kbp in length and encode 150–300 proteins. Replication occurs in the cytoplasm of infected cells in regions known as viral factories, where immature and mature particles can be observed. Some mature particles acquire an envelope near the Golgi complex and are subsequently released from the cell, whereas nonenveloped particles are released by cell lysis (FAUQUET et al., 2005; FENNER et al., 1992).

Transmission may occur through the bites of hematophagous insects or contact with contaminated food, water, secretions, and fomites. The virus cannot penetrate intact skin and requires cuts or abrasions in the skin or mucosa to establish infection (CUBAS & GODOY, 2004). The disease may occur in cutaneous, diphtheritic, or mixed forms, depending on strain virulence, lesion distribution, and host susceptibility. The cutaneous form, also termed dry pox and probably the most common form, is characterized by nodular, proliferative, or crusted wart-like lesions, mainly in featherless areas such as the legs, feet, beak, and periocular region. Mild lesions are generally small and cause little debilitation; however, extensive lesions can impair vision, feeding, and breathing. Lesions in feathered areas, especially subtle ones, may also go unnoticed. In the diphtheritic form, or wet pox, proliferative plaques or membranes develop on the mucosa of the oral cavity and pharynx and sometimes the larynx and trachea, potentially causing respiratory difficulty, emaciation, and death. Cutaneous and diphtheritic lesions may occur simultaneously, constituting the mixed presentation (HOFSTAD et al., 1988; WILLIAMS et al., 2021).

Histologically, lesions may show epithelial hyperplasia and hypertrophy, hydropic degeneration, and eosinophilic intracytoplasmic inclusion bodies, known as Bollinger bodies, in skin or mucosal cells (GREENACRE, 2005).

In a study conducted at Tietê Ecological Park during an outbreak involving birds seized from illegal trade, Catroxo et al. (2009) reported avian pox in seven red-cowled cardinals (Paroaria dominicana), as well as other passerine species. All examined birds had lesions, and typical poxvirus particles were demonstrated by transmission electron microscopy using negative staining. Ultrathin skin sections revealed type A, type B, or Bollinger intracytoplasmic inclusions and fibrillar inclusions, complete and incomplete particles, and deformed nuclei with marginalized chromatin.

Transmission electron microscopy is particularly suitable for rapid identification of infectious agents. Negative staining reveals the morphology and dimensions of viral particles, assisting in their identification and classification. In addition to being rapid, it requires only a small sample and enables the detection of different viral particles in a single preparation (DOANE & ANDERSON, 1987; ALMEIDA, 1983; HAZELTON & GELDERBLOM, 2003).

Immunoelectron microscopy may be used when the number of viral particles in a sample is low, enabling identification through both morphology and the specific antigen–antibody reaction (KATZ & KLOHN, 1984). Colloidal gold immunolabeling enables detection and localization of viral antigens in infected cells and assists in identifying strains and antigenic variants (KJELDSBERG, 1986; PATTERSON & OXFORD, 1986).

Conclusion

This is the first record of avian pox in the red-crested cardinal (Paroaria coronata) and the second in the red-cowled cardinal (Paroaria dominicana). The combination of histopathological findings, visualization of viral particles by negative staining and in ultrathin sections, and colloidal gold immunolabeling confirmed the presence of poxvirus and demonstrated different stages of its morphogenesis in cutaneous lesions.

The results show that wild birds are also susceptible to infectious diseases capable of impairing mobility, feeding, and overall condition. This finding is particularly important for wildlife conservation, because diseases such as avian pox may increase the vulnerability of wild populations, including species threatened with extinction, especially when associated with animal trafficking, crowding, stress, inadequate husbandry, and the presence of vectors. Accordingly, early diagnosis, quarantine, isolation of affected birds, biosafety measures, and insect control are essential in wildlife screening and recovery centers.

Agriculture Journal IJOEAR Call for Papers

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