Unusual pox lesions found in Chinese jungle mynahs (Acridotheres cristatellus)

Abstract

Pox lesions involving feathered and unfeathered skin, the oral cavity and the uropygial gland were found in Chinese jungle mynahs. Characteristic intracytoplasmic inclusions were detected in the proliferative cells of all lesions. Ultrastructurally, the virus particles consisted of a convoluted outer membrane enclosing lateral bodies and a biconcave central core, typical for poxvirus. The nucleotide sequences of the amplicon obtained with a set of primers for the 4b core protein of fowl poxvirus revealed that the mynah poxvirus was phylogenetically related to wood pigeon poxvirus. This is the first report of poxvirus infection affecting the uropygial gland.

Lésions varioliques inhabituelles, observées chez un martin huppé chinois (Acridotheres cristatellus)

Les lésions varioliques, comprenant la peau couverte ou non de plumes, la cavité orale et la glande uropygienne, ont été observées chez des martins huppés chinois. Des inclusions intra cytoplasmiques caractéristiques ont été notées dans les cellules prolifératives de toutes les lésions. Sur le plan ultra structural, les particules virales se présentaient sous forme de membrane externe enfermant les corps latéraux et d'un noyau central biconcave, typiques des poxvirus. En se basant sur les séquences nucléotidiques de l'amplicon obtenu avec un set d'amorces pour la protéine 4b de noyau de la variole aviaire, il est apparu que le virus de la variole du martin était proche phylogénétiquement de celui du pigeon ramier. C'est le premier rapport d'une infection affectant la glande uropygienne.

Ungewöhnliche Pockenläsionen in chinesischen Dschungelmainas (Acridotheres cristatellus)

In chinesischen Dschungelmainas wurden Pockenläsionen in der befiederten und unbefiederten Haut, der Schnabelhöhle und der Bürzeldrüse gefunden. In allen Läsionen wurden in den proliferativen Zellen charakteristische intrazytoplasmatische Einschlusskörperchen nachgewiesen. Elektronenmikroskopisch konnten für Pockenviren typische Viruspartikel mit gewundener Außenhülle, die die Lateralkörper und den bikonkaven zentralen Kern umschließt, festgestellt werden. Basierend auf der Nukleotidsequenz des Amplikons, das mittels des Primersets für das 4b-Coreprotein des Geflügelpockenvirus gewonnen wurde, erwies sich das Maina-Pockenvirus als phylogenetisch verwandt mit den Ringeltaubenpockenvirus. Dies ist die Erstbeschreibung einer Pockenvirusinfektion, die die Bürzeldrüse befällt.

Lesiones inusuales de viruela aviar en pájaros minah (Acridotheres cristatellus)

Se describen lesiones de viruela aviar en piel con y sin plumas, en cavidad oral y en la glándula uropígea de pájaros minah. Se observaron los cuerpos de inclusión intracitoplasmáticos en las células que proliferaban en todas las lesiones. Ultrastructuralmente, las partículas víricas consistieron en una membrana externa replegada que contenía los cuerpos laterales y un núcleo central bicóncavo, típico de poxvirus. En base a la secuencia nucleotídica del amplicón obtenido con un par de cebadores que amplificaban la proteína 4b del núcleo del poxvirus aviar se observó que el poxvirus del minah se encontraba filogenéticamente relacionado con el poxvirus de la paloma torcaz. Esta es la primera descripción de una infección por poxvirus que afecta la glándula uropígea.

Introduction

Avian pox has been widely reported in domestic and various wild birds (Ritchie, 1995).

It induces a disease characterized by proliferative lesions on the unfeathered skin, and diphtheritic lesions in the upper respiratory and digestive tracts (Riddell, 1987). In addition, lesions have been reported in the lung of canaries (Harrison & Harrison, 1986) and on the cranium of lovebirds (Tsai et al., 1997).

Mynah birds are a common pet bird; however, diseases of mynahs are not as extensively documented as those of psittacine birds (Panigraphy & Senne, 1991; Lüschow et al., 2004; Weli et al., 2004). Pox lesions on the eyelids, around the beak and over the skin of the head and neck have been found in Hill, Bali and Rothchild mynahs (Karpinski & Clubb, 1986). The poxvirus that infects free-ranging starlings is suggested to be infectious for mynah birds but non-pathogenic for domestic fowl (Landolt & Kocan, 1976).

This report descries poxvirus infection of Chinese jungle mynahs, and is the first report of pox lesions in the uropygial gland of birds. It also describes molecular characterization of the virus.

Case Report

Two hundred adult Chinese jungle mynahs (Acridotheres cristatellus) were kept in large outdoor flight cages on a breeding farm in southern Taiwan. Two birds died, and were presented for necropsy. No vaccinations or treatments had been administered to the birds on the farm.

The carcasses were emaciated. One mynah had whitish proliferative nodules under the base of the lower jaw and at the angle of the beak. Several yellowish-white nodules, some of which were ulcerated, were visible on the inner mucosa of the upper and lower beaks when the mouth was opened (Figure 1). Striking lesions were found in the uropygial gland, where a greyish-white tumour-like mass, about 1 cm in diameter, with firm consistency was protruding (Figure 2). The feather follicles neighbouring the uropygial mass had nodular lesions, and whitish proliferative patches on the unfeathered skin. The other bird had a smaller uropygial mass, about 0.8 cm in diameter, but there were no oral or skin lesions.

Figure 1.  Several rounded pox lesions with or without ulceration (arrows) are found on the oral mucosa.

Figure 2.  A large greyish-white nodule is protruding from the rump.

Microscopic examination of the lesions revealed marked hyperplasia and ballooning degeneration of epidermal cells of the skin and epithelial cells of the mouth and uropygial gland, associated with formation of intracytoplasmic eosinophilic inclusion bodies encircled by a clear zone. Lesions were present on the unfeathered and feathered skin, involving both the epidermis and pulp of feather follicles. Similar histological changes were found on the oral mucosa. Both mynahs had typical pox lesions in the uropygial glands (Figure 3a,b). The glandular acini had two types of changes. One was loss of most normal acinar epithelial cells, with replacement by proliferating epithelial cells. Within their cytoplasms were eosinophilic, irregularly rounded, intracytoplasmic inclusion bodies. The other type contained only a small amount of necrotic cell debris in the remaining acini. There was no inflammatory reaction. In addition, both mynahs had mycotic ventriculitis in the gizzard. This was characterized by marked hyperplasia of the koilin layer and invasion of pseudomycelia and blastospores, typical for Candida spp.

Figure 3.  (a) The normal acinar cells of the uropygial gland are replaced by adenomatoid proliferation cells (haematoxylin and eosin, bar = 100 µm), (3b) The proliferating cells show vacuolar degeneration and contain irregular cytoplasmic inclusions) (haematoxylin and eosin, bar = 20 µm), (3c) Electron micrograph of a cytoplasmic inclusion from an affected uropygial gland containing many poxvirus particles (bar = 1 µm).

Electron microscopic examination of the formalin-fixed tissues with pox-like lesions revealed virus particles consisting of a convoluted outer membrane enclosing lateral bodies and a biconcave central core (Figure 3c). The morphology and size of the particles was consistent with poxvirus.

Portions of pooled uropygial glands were minced and homogenized in phosphate-buffered saline to make a 5% homogenate. Extraction of viral DNA from the homogenate was performed using a commercial kit (VIOGENE; Fu-Lein Co., Taiwan) based on proteinase K digestion. Purified viral DNA was amplified by polymerase chain reaction (PCR) using a commercial kit (Extensor Hi-Fidelity PCR Enzyme, AB-0720; ABgene Co., UK). The PCR for poxvirus DNA was performed according to a previously described method (Lee & Lee, 1997). It was used to amplify a 578-base-pair fragment of fowl poxvirus genome and with a set of primers framing a region within the gene coding for 4b core protein.

The nucleotide sequences of the 4b core protein of poxvirus were aligned using a Clustal method with the PAM250 residue weight table of DNASTAR software (DNASTAR Inc., Madison, Wisconsin, USA). The amplified product from our case showed 95.8% homology to wood pigeon poxvirus and less than 83.9% with other poxviruses, such as sparrow, stone curlew, canary, great tit, falcon, ostrich, pigeon, turkey and fowl (Table 1) (Lüschow et al., 2004; Weli et al., 2004). The topology of the tree revealed that our mynah poxvirus was phylogenetically most closely related to wood pigeon poxvirus (Figure 4).

Figure 4.  Phylogenetic tree based on the nucleotide sequence between positions 1 and 143 of the 4b gene. The tree is constructed using a Clustal method with the PAM250 residue weight table of DNASTAR software.

Table 1.  Pair-wise sequence comparison of the nucleotide of the 4b gene using a Clustal method with the PAM250 residue weight table of DNASTAR software

The uropygial gland is often overlooked during routine physical examination of birds. Diseases of this gland are rarely reported. The most common abnormality is vitamin A deficiency (Bauck et al., 1997). Neoplasia including adenomas, squamous cell carcinomas, papillomas and adenocarcinomas have been described. However, infectious adenitis is rare except in psitttacine beak and feather disease, or frequently secondary to hypovitaminosis A or immunosuppressive disease.

The pathological and electron microscopic findings and PCR results strongly supported a diagnosis of poxvirus infection in these Chinese jungle mynahs. In mynahs, poxvirus induces proliferative lesions around the beak and the eyes in field cases, and on the eyelids, in the oropharynx and on the traumatized areas of face and limbs in experimentally inoculated birds (Panigraphy & Senne, 1991). Poxvirus infection of mynahs has never been found before in Taiwan. One of our cases showed the typical pox lesions on the skin and the oral mucosa, but interestingly the uropygial gland was also affected. Pox lesions in the uropygial gland have not previously been reported in birds. Phylogenetically, the virus was closely related to wood pigeon poxvirus. The origin and transmission route of the virus is unknown.

The emaciated condition of the mynahs might have resulted from difficulty in pecking and swallowing food because of pox lesions involving the oral cavity and the angle of the beak (one bird), as well as the malfunction of the gizzard due to mycotic ventriculitis (both birds). Mycotic infection of the gizzard is often associated with immunocompromise in birds. However, there was no evidence for the condition in the present study. The normal bacterial flora of gastrointestinal mucosal surfaces have an inhibitory effect on the growth of Candida species. Therefore, suppression of normal flora by routinely prolonged use of oxytetracycline in the feed as a growth promoter might have been the predisposing factor for fungal propagation.

Translations of the abstract in French, Germany and Spanish are available on the Avian Pathology website.