Respiratory Herpesvirus Infection in Two Indian Ringneck Parakeets Tatjana Lazic Iowa State University

Veterinary Pathology Publications and Papers Veterinary Pathology

3-2008 Respiratory herpesvirus infection in two Indian Ringneck parakeets Tatjana Lazic Iowa State University

Mark R. Ackermann Iowa State University, [email protected]

Jo M. Drahos Riverside Animal Hospital

Judith Stasko United States Department of Agriculture

Joseph S. Haynes Iowa State University, [email protected]

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Abstract A flock of ndI ian Ringneck parakeets (Psittacula krameri manillensis) was imported to the United States from Australia. Soon after, 1 parakeet suddenly died, and a second parakeet died after a 2-day course of illness, which consisted of anorexia, lethargy, emaciation, and dyspnea. At necropsy, the affected birds had diffuse consolidation and red discoloration of the lungs, as well as thickened, congested air sacs. The microscopic examination revealed multifocal, necrotizing bronchitis, parabronchitis, and interstitial pneumonia. The lumen of the affected airways contained numerous, large syncytial cells with up to 15 nuclei. The uclein of these syncytial cells often contained large, eosinophilic inclusion bodies, consistent with herpesvirus. The epithelium of the trachea and air sacs was hypertrophied and contained syncytial cells with intranuclear inclusion bodies similar to the bronchi. In addition, a few intranuclear inclusion bodies were also present in the epithelial cells that line the air capillaries. On ultrastructural examination, the nuclei of degenerating epithelial cells contained clusters of viral nucleocapsid proteins and unenveloped, icosahedral, viral particles that were approximately 90 nm in diameter. In addition, some epithelial cells contained clusters of enveloped viral particles approximately 105 nm in diameter, within the cytocavitary network. These lesions are characteristic of those caused by respiratory herpesvirus of parakeets.

Keywords Herpesvirus, parakeet, Psittacid, respiratory

Disciplines Veterinary Pathology and Pathobiology

Comments This article is from Journal of Veterinary Diagnostic Investigation 20, no. 2 (March 2008): 235–238, doi:10.1177/104063870802000217.

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This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/vpath_pubs/20 Journal of Veterinary Diagnostic Investigation http://vdi.sagepub.com/

Respiratory herpesvirus infection in two Indian Ringneck parakeets Tatjana Lazic, Mark R. Ackermann, Jo M. Drahos, Judith Stasko and Joseph S. Haynes J VET Diagn Invest 2008 20: 235 DOI: 10.1177/104063870802000217

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J Vet Diagn Invest 20:235–238 (2008)

Respiratory herpesvirus infection in two Indian Ringneck parakeets

Tatjana Lazic,1 Mark R. Ackermann, Jo M. Drahos, Judith Stasko, Joseph S. Haynes

Abstract. A flock of Indian Ringneck parakeets (Psittacula krameri manillensis) was imported to the United States from Australia. Soon after, 1 parakeet suddenly died, and a second parakeet died after a 2-day course of illness, which consisted of anorexia, lethargy, emaciation, and dyspnea. At necropsy, the affected birds had diffuse consolidation and red discoloration of the lungs, as well as thickened, congested air sacs. The microscopic examination revealed multifocal, necrotizing bronchitis, parabronchitis, and interstitial pneumonia. The lumen of the affected airways contained numerous, large syncytial cells with up to 15 nuclei. The nuclei of these syncytial cells often contained large, eosinophilic inclusion bodies, consistent with herpesvirus. The epithelium of the trachea and air sacs was hypertrophied and contained syncytial cells with intranuclear inclusion bodies similar to the bronchi. In addition, a few intranuclear inclusion bodies were also present in the epithelial cells that line the air capillaries. On ultrastructural examination, the nuclei of degenerating epithelial cells contained clusters of viral nucleocapsid proteins and unenveloped, icosahedral, viral particles that were approximately 90 nm in diameter. In addition, some epithelial cells contained clusters of enveloped viral particles approximately 105 nm in diameter, within the cytocavitary network. These lesions are characteristic of those caused by respiratory herpesvirus of parakeets. Key words: Herpesvirus; parakeet; Psittacid; respiratory.

From the Department of Veterinary Pathology, Iowa State University, Ames, IA (Lazic, Ackermann, Haynes), the Riverside Animal Hospital, Muscatine, IA (Drahos), and the National Animal Disease Center, Ames, IA (Stasko). 1Corresponding Author: Tatjana Lazic, Department of Veterinary Pathology, Vet Med Building, Iowa State University, Ames, IA 50011. [email protected]

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Herpesviruses comprise a large family of enveloped DNA viruses. They have a wide variety of hosts, and nearly every mammalian and avian species is a natural host of one or more herpesvirus strains. This report documents a novel lower-respiratory-tract disease in 2 Indian Ring- neck parakeets caused by a herpesvirus. Tissues from 2 Indian Ringneck parakeets from a flock imported to the United States from Australia were submitted to the Veterinary Pathology Department of Iowa State University (Ames, IA) for diagnostic evaluation. The first bird was presented dead to the referring veterinarian. The second bird was presented with lethargy, emaciation, and dyspnea, and died after a 2-day course of illness. The trachea, lung, air sacs, and liver were collected at necropsy and were fixed in 10% neutral-buffered formalin. According to the referring veterinarian, the gross lesions in the first bird consisted of the markedly and diffusely thickened air sacs, heavy and diffusely red lungs, and disseminated pale foci in the liver. The second parakeet Figure 1. Syncytial cell (arrow) with intranuclear inclusion bodies (open arrows). had similar lung and air sac lesions, with no hepatic involvement. Tissues received by the referring veterinarian were routinely processed, embedded in paraffin, and 5-mm Herpesvirus subfamilies include alpha-herpesviruses, thick sections were stained with hematoxylin and eosin. beta-herpesviruses, and gamma-herpesviruses. Alpha-her- By light microscopy, the lung and airway sacs of both pesviruses are associated with rapid viral replication, host parakeets had similar lesions. The airway lumina, airway cell lysis, and the ability to establish latent infection.5 An epithelial lining, and air capillaries contained numerous, example of avian alpha-herpesvirus is Gallid herpesvirus 1 large syncytial cells. The syncytial cells contained up to 15 (family Herpesviridae, subfamily Alphaherpesvirinae, genus identifiable nuclei, with occasional eosinophilic and intra- Iltovirus), commonly known as Infectious laryngotracheitis nuclear inclusion bodies, both of which are often associated virus (ILTV) of chickens, which causes upper-respiratory- with foci of necrosis (Fig. 1). The airway epithelium was tract infection manifested as necrotizing pharyngitis, multifocally eroded or lined by hypertrophied cells, and the laryngitis, tracheitis, and, occasionally, mild pneumonia.23 airway lumina often contained mucus, necrotic cell debris, and heterophils. There were scattered foci of congestion and hemorrhage throughout the pulmonary parenchyma. The epithelium of air sacs and the trachea contained syncytial cells similar to the lung airways. The lung tissue of the first bird contained numerous fungal hyphae, which extended through the mucosa of the large airways into the adjacent parenchyma. The fungal hyphae had parallel walls, septa 5–10 mmindiameter,anddichotomous branching, which is most consistent with Aspergillus sp. In addition, the liver of the first parakeet contained scattered foci of coagulative necrosis but lacked syncytial cells or intranuclear inclusion bodies. The second bird did not contain any identifiable hepatic lesions. Specimens of lung and air sac from the second bird were further submitted to the National Animal Disease Center (Ames, IA) for electron transmission microscopic evaluation. The tissues were routinely processed, embedded, and stained with lead citrate and uranyl acetate. On ultrastruc- ture examination, the nuclei of degenerating epithelial cells contained clusters of viral nucleocapsid proteins and unenveloped, icosahedral viral particles that were approximately 90 nm in diameter (Fig. 2). In addition, some epithelial cells contained clusters of enveloped viral particles approximately 105 nm in diameter, within the cytocavitary network (Fig. 3). These features are characteristic of herpesviruses, thus the diagnosis was necrotizing Figure 2. The intranuclear viral particles are not yet bronchitis, bronchiolitis, and air sacculitis because of enveloped and, therefore, are approximately 95 nm in diameter infection with herpesvirus. (open arrows).

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liver of the first bird lacked syncytial cells and intranuclear inclusion bodies. Based on the similarity of the lesions, the authors believe that the herpesvirus in the present case is very similar to, if not the same as, the respiratory herpesvirus of the 2 parakeets previously reported in the United States9 and Japan.26 The histological lesions in the lung of the Bourke’s parakeet included segmental necrosis, cuboidal metaplasia, and syncytial giant cells in the parabronchial epithelium. On transmission electron microscopy, the location, morphology, and size of the viral particles are typical of a herpesvirus. No histological lesions were found in the liver. The report from Japan describes disease in 14 parakeets that died of herpesvirus-induced pneumonia and air sacculitis.9 The histological lesions included necrotizing tracheitis, pneumonia, and air sacculitis, with syncytial cells and herpes-like inclusion bodies in the lung. Many affected birds had secondary pulmonary aspergillosis, chlamydiosis, or candidiasis. Because of its respiratory tropism, it is speculated that this virus may be a variant of ILTV in chickens or ATV in Psittaciformes. However, both of those viruses primarily have tropism for the upper respiratory tract manifested as necrotizing pharyngitis, laryngitis, tracheitis, and occa- Figure 3. Electron micrograph of epithelial cell. The cyto- sional pneumonia. In addition, pneumonia is characterized plasm contains a cluster of enveloped viral particles within by epithelial cell hyperplasia, edema, and airway mucosal cytocavitary network, which approximately measure 105 nm in infiltration by moderate numbers of mixed inflammatory diameter (open arrow). populations, without syncytial cell formation. Another possibility is that this lower respiratory disease In Psittaciformes, there are several recognized diseases is an unusual manifestation of PD. Pacheco’s disease associated with avian alpha-herpesviruses. Infection with occurs worldwide,1,3,4,6,10,12,13 and once recognized, it was Psittacid herpesvirus 1 (PsHV-1), formerly known as speculated that PD is caused by a single PsHV serotype. Pacheco’s disease (PD), is characterized by massive hepatic 25 18 Today, there are at least 5 different PsHV serotypes necrosis with formation of syncytial cells. Amazon recovered from Psittacid birds, with serotype 1 (PsHV-1) tracheitis virus (ATV) is a cause of upper-respiratory-tract 24 18 being the most common. Pacheco’s disease is primarily lesions similar to ILTV in chickens. There are several characterized by a massive hepatic necrosis,14 variably other herpesviruses associated with more chronic, non–life- accompanied by necrotizing lesions in intestines, spleen, threatening skin and/or mucosal lesions. It is speculated kidney, pancreas, and endocrine glands;17,18 there have that the feather abnormalities referred to as ‘‘feather been infrequent reports of unusual manifestations of PD dusters’’ in European Budgerigars are caused by a herpes- characterized by lung15,18,20 lesions, which would imply 18 virus. The isolated virus is unrelated to those strains a possible variation in PsHV tissue tropism. In another found in psittacine birds, and the significance of its report, an unusual outbreak of necrotizing esophagitis2 occurrence in these feather lesions remains to be de- occurred in Pionus sp. and Amazon parrots. The liver termined. There are reports of possible herpesvirus in- contained only focal areas of necrosis with rare intra- volvement of wart-like skin lesions on the feet of cockatoos nuclear inclusion bodies. In the current study, the authors 18 and macaws; however, this remains to be confirmed. speculate that the esophageal and hepatic lesions might be Recently, a novel Psittacid herpesvirus strain was isolated a manifestation of PD caused by serologically distinct from the mucosal papillomas of neotropical parrots21 and variant of PsHV. Herpesviruses have been isolated from from cloacal and cutaneous papillomas of African grey many other bird orders, including Columbiformes (pigeons parrots.22 and doves),11,19 Falconiformes (falcons),7 and Anseri- However, there have been reports of a different herpes- formes (ducks).8 In these birds, the herpesvirus-associated virus of parakeets that has tropism for the lower respiratory lesions were mainly localized within the gastrointestinal tract, with no hepatic or significant upper-respiratory-tract tract. However, in Passeriformes (songbirds), the herpesvi- involvement. One was from the United States,9 in rus lesions were confined to the conjunctiva and the a Bourke’s parakeet, and the other was from Japan.26 This respiratory tract.16 Upon DNA sequencing, this virus was virus is referred to as ‘‘respiratory herpesvirus of para- related to ILTV in gallids. keets’’ and represents an unusual manifestation of herpes- By considering the distribution and characteristics of the virus-induced disease in parakeets. In these 2 parakeets, the lesions, as well as electron microscopy findings, it is herpes-like inclusion bodies were identified within epithelial hypothesized that this virus is very similar to the virus cells and syncytial cells of the trachea, bronchi, para- previously reported in Japan9 and the United States.26 bronchi, air capillaries, and air sacs. The necrotic foci in the Although there are several publications that reported the

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